Prion diseases are fatal neurodegenerative disorders caused by an aggregated form of the prion protein, PrPSc. Prions are the only protein aggregates that are naturally transmitted as an infectious disease, and most recently, human to human transmission likely occurred by transfusion of prion-contaminated blood. Most cases of natural transmission occur through a peripheral exposure followed by prion spread to the CNS. Although prions are thought to spread via peripheral nerves to the CNS, the molecular mechanisms underlying neuronal transit are unclear. For this reason, a major goal of this grant application is to understand how prion aggregates spread to the CNS. We hypothesize that prions propagate by retrograde axonal transport and will address this hypothesis using in vitro and in vivo experimental models. To accomplish this goal, three aims are proposed. In the first aim, we will test the mechanism of PrPSc axonal transport using compartmentalized neuronal cultures that have a liquid separation between the axon terminals and the cell bodies. We will additionally assess the peripheral nerve transport of prion strains in vivo. In the second aim, we will measure the structural properties of the highly virulent prion strains that readily spread to he CNS. In the third aim, we will determine the role of the GPI membrane anchor of PrPC on prion spread to the brain using transgenic mice and primary neurons that express PrPC possessing or lacking the GPI anchor. We expect that these studies will provide critical missing information on the basic mechanisms of prion spread that will be essential for the development of immunotherapies and novel therapeutic strategies to prevent or arrest disease progression.